Structure and function of placental growth factor

Placental growth factor (PlGF) belongs to the same family as the vascular endothelial growth factor A (VEGF-A). Recent gene inactivation studies in mice have demonstrated that loss of PlGF does not affect development, reproduction, or normal postnatal life. However, the mice show significantly impaired angiogenesis and arteriogenesis during pathological conditions such as ischemia and tumor formation, conditions in which the expression of VEGF-A is normally increased. Mice expressing a truncated form of the specific receptor for PlGF, the VEGF receptor 1 (VEGFR-1), show impaired angiogenesis similar to that observed in Plgf(-/-)mice. These data suggest a pivotal role for PlGF and VEGFR-1 in regulating VEGF-A-dependent angiogenesis under pathological conditions. VEGF-A has been utilized for the therapeutic stimulation of new blood vessels in ischemic hearts and limbs, with controversial results from the initial clinical experience. This review discusses the possibility of using the PlGF/VEGFR-1 pathway as an alternative target for angiogenic therapy.     

RETRACTED ARTICLE: Gold nanoparticles inhibit vascular endothelial growth factor-induced angiogenesis and vascular permeability via Src dependent pathway in retinal endothelial cells

The purpose of this study was to investigate the effect of gold nanoparticles on the signaling cascade related to angiogenesis and vascular permeability induced by Vascular Endothelial Growth Factor (VEGF) in Bovine retinal endothelial cells (BRECs). The effect of VEGF and gold nanoparticles on cell viability, migration and tubule formation was assessed. PP2 (Src Tyrosine Kinase inhibitor) was used as the positive control and the inhibitor assay was performed to compare the effect of AuNPs on VEGF induced angiogenesis. The transient transfection assay was performed to study the VEGFR2/Src activity during experimental conditions and was confirmed using western blot analysis. Treatment of BRECs with VEGF significantly increased the cell proliferation, migration and tube formation. Furthermore, gold nanoparticles (500 nM) significantly inhibited the proliferation, migration and tube formation, in the presence of VEGF in BRECs. The gold nanoparticles also inhibited VEGF induced Src phosphorylation through which their mode of action in inhibiting angiogenic pathways is revealed. The fate of the gold nanoparticles within the cells is being analyzed using the TEM images obtained. The potential of AuNPs to inhibit the VEGF165-induced VEGFR-2 phosphorylation is also being confirmed through the receptor assay which elucidates one of the possible mechanism by which AuNPs inhibit VEGF induced angiogenesis. These results indicate that gold nanoparticles can block VEGF activation of important signaling pathways, specifically Src in BRECs and hence modulation of these pathways may contribute to gold nanoparticles ability to block VEGF-induced retinal neovascularization.     

Involvement of VEGFR-2 (kdr/flk-1) but not VEGFR-1 (flt-1) in VEGF-A and VEGF-C-induced tube formation by human microvascular endothelial cells in fibrin matrices in vitro

Different forms of vascular endothelial growth factor (VEGF) and their cellular receptors (VEGFR) are associated with angiogenesis, as demonstrated by the lethality of VEGF-A, VEGFR-1 or VEGFR-2 knockout mice. Here we have used an in vitro angiogenesis model, consisting of human microvascular endothelial cells (hMVEC) cultured on three-dimensional (3D) fibrin matrices to investigate the roles of VEGFR-1 and VEGFR-2 in the process of VEGF-A and VEGF-C-induced tube formation. Soluble VEGFR-1 completely inhibited the tube formation induced by the combination of VEGF-A and TNFα (VEGF-A/TNFα). This inhibition was not observed when tube formation was induced by VEGF-C/TNFα or bFGF/TNFα. Blocking monoclonal antibodies specific for VEGFR-2, but not antibodies specifically blocking VEGFR-1, were able to inhibit the VEGF-A/TNFα-induced as well as the VEGF-C/TNFα-induced tube formation in vitro. PlGF-2, which interacts only with VEGFR-1, neither induced tube formation in combination with TNFα, nor inhibited or stimulated by itself the VEGF-A/TNFα-induced tube formation in vitro. These data indicate that VEGF-A or VEGF-C activation of the VEGFR-2, and not of VEGFR-1, is involved in the formation of capillary-like tubular structures of hMVEC in 3D fibrin matrices used as a model of repair-associated or pathological angiogenesis in vitro. This revised version was published online in June 2006 with corrections to the Cover Date.     

Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1): a dual regulator for angiogenesis

Vascular endothelial growth factor receptor-1 (VEGFR-1) is a member of the VEGFR family, and binds VEGF-A, PlGF, and VEGF-B. An important feature of VEGFR-1 is that, unlike other VEGFR genes, it expresses two types of mRNA, one for a full-length receptor and another for a soluble short protein known as soluble VEGFR-1 (sFlt-1). The binding-affinity of VEGFR-1 for VEGF-A is one order of magnitude higher than that of VEGFR-2, whereas the kinase activity of VEGFR-1 is about 10-fold weaker than that of VEGFR-2. Through its ligand-binding region and by trapping ligands, VEGFR-1 plays a negative role in angiogenesis at embryogenesis. In adulthood, however, VEGFR-1 is expressed not only on endothelial cells but also on macrophages, and promotes the function of macrophages, inflammatory diseases, cancer metastasis, and atherosclerosis via its kinase activity. Soluble VEGFR-1 is abnormally overexpressed in the placenta of preeclamptic patients, and suggested to cause the major pathological symptoms on the maternal side such as hypertension and renal dysfunction, most likely by blocking the physiological VEGF-A. VEGFR-1 including its soluble form is involved in a variety of human illnesses, making it an important target in the development of new strategies to suppress disease.     

Overexpression of the soluble vascular endothelial growth factor receptor in preeclamptic patients: pathophysiological consequences

Several growth factors such as vascular endothelial growth factor (VEGF)-A and placental growth factor (PlGF) are involved in the placental vascular development. We investigated whether dysregulation in the VEGF family may explain the defective uteroplacental vascularization characterizing preeclampsia. We compared pregnancies complicated by early onset severe preeclampsia or intrauterine growth retardation to normal pregnancies. Maternal plasma, placentas, and placental bed biopsies were collected. The mRNA levels of VEGF-A, PlGF, and their receptors were quantified in placentas and placental beds. Levels of VEGF-A, PlGF, and soluble VEGF receptor (VEGFR) were assessed in maternal plasma. In compromised pregnancies, elevated levels of VEGF-A and VEGFR-1 mRNAs may reflect the hypoxic status of the placenta. On contrast, the membrane-bound VEGFR-1 was decreased in the placental bed of preeclamptic patients. Preeclampsia was associated with low levels of circulating PlGF and increased levels of total VEGF-A and soluble VEGFR-1. Free VEGF-A was undetectable in maternal blood. Immunohistochemical studies revealed that VEGF-A and PlGF were localized in trophoblastic cells. Altogether, our results suggest two different pathophysiological mechanisms associated with preeclampsia. The first one is related to an overproduction of competitive soluble VEGFR-1 that may lead to suppression of VEGF-A and PlGF effects. The second one is the down-regulation of its membrane bound form (VEGFR-1) in the placental bed, which may result in the defective uteroplacental development.     

Urokinase receptor expression on human microvascular endothelial cells is increased by hypoxia: implications for capillary-like tube formation in a fibrin matrix

Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix. hMVECs stimulated with fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor (VEGF) together with tumor necrosis factor-alpha (TNF-alpha) formed 2- to 3-fold more tubular structures under hypoxic conditions than in normoxic (20% oxygen) conditions. In both conditions the in-growth of capillary-like tubular structures into fibrin required cell-bound urokinase-type plasminogen activator (uPA) and plasmin activities. The hypoxia-induced increase in tube formation was accompanied by a decrease in uPA accumulation in the conditioned medium. This decrease in uPA level was completely abolished by uPA receptor-blocking antibodies. During hypoxic culturing uPA receptor activity and messenger RNA (mRNA) were indeed increased. This increase and, as a consequence, an increase in plasmin formation contribute to the hypoxia-induced stimulation of tube formation. A possible contribution of VEGF-A to the increased formation under hypoxic conditions is unlikely because there was no increased VEGF-A expression detected under hypoxic conditions, and the hypoxia-induced tube formation by FGF-2 and TNF-alpha was not inhibited by soluble VEGFR-1 (sVEGFR-1), or by antibodies blocking VEGFR-2. Furthermore, although the alpha(v)-integrin subunit was enhanced by hypoxia, blocking antibodies against alpha(v)beta(3)- and alpha(v)beta(5)-integrins had no effect on hypoxia-induced tube formation. Hypoxia increases uPA association and the angiogenic response of human endothelial cells in a fibrin matrix; the increase in the uPA receptor is an important determinant in this process. (Blood. 2000;96:2775-2783)     

Tissue deformation spatially modulates VEGF signaling and angiogenesis

Physical forces play a major role in the organization of developing tissues. During vascular development, physical forces originating from a fluid phase or from cells pulling on their environment can alter cellular signaling and the behavior of cells. Here, we observe how tissue deformation spatially modulates angiogenic signals and angiogenesis. Using soft lithographic templates, we assemble three-dimensional, geometric tissues. The tissues contract autonomously, change shape stereotypically and form patterns of vascular structures in regions of high deformations. We show that this emergence correlates with the formation of a long-range gradient of Vascular Endothelial Growth Factor (VEGF) in interstitial cells, the local overexpression of the corresponding receptor VEGF receptor 2 (VEGFR-2) and local differences in endothelial cells proliferation. We suggest that tissue contractility and deformation can induce the formation of gradients of angiogenic microenvironments which could contribute to the long-range patterning of the vascular system.     

Redundant roles of VEGF-B and PlGF during selective VEGF-A blockade in mice

Vascular endothelial growth factor-A (VEGF-A) and its 2 transmembrane tyrosine-kinase receptors, VEGFR-1 and VEGFR-2, constitute a ligand-receptor signaling system that is crucial for developmental angiogenesis. VEGF-B and placental growth factor (PlGF) activate VEGFR-1 selectively, however, mice lacking either ligand display only minor developmental defects. We hypothesized that the relative contributions of VEGF-B and PlGF to VEGFR-1 signaling may be masked in the presence of VEGF-A, which is abundantly expressed during postnatal development. To test this hypothesis, neonatal or adult mice were treated with a monoclonal antibody (G6-23-IgG) blocking murine VEGF-A or a soluble VEGFR-1 receptor IgG chimeric construct [mFlt(1-3)-IgG], which neutralizes VEGF-A, VEGF-B, and PlGF. Both compounds attenuated growth and survival of neonatal mice to similar extents and the pathophysiologic alterations, including a reduction in organ size and vascularization, changes in gene expression, and hematologic end points, were essentially indistinguishable. In adult mice, we observed only minor changes in response to treatment, which were similar between both anti-VEGF compounds. In conclusion, our findings suggest that PlGF and VEGF-B do not compensate during conditions of VEGF-A blockade, suggesting a minor role for compensatory VEGFR-1 signaling during postnatal development and vascular homeostasis in adults. The absence of compensatory VEGFR-1 signaling by VEGF-B and PlGF may have important implications for the development of anticancer strategies targeting the VEGF ligand/receptor system.     

c-ABL modulates MAP kinases activation downstream of VEGFR-2 signaling by direct phosphorylation of the adaptor proteins GRB2 and NCK1

Vascular Endothelial Growth Factor-A (VEGF-A) is a key molecule in normal and tumor angiogenesis. This study addresses the role of c-ABL as a novel downstream target of VEGF-A in primary Human Umbilical Vein Endothelial Cells (HUVEC). On the basis of immunoprecipitation experiments, in vitro kinase assay and RNA interference, we demonstrate that VEGF-A induces the c-ABL kinase activity through the VEGF Receptor-2/Phosphatidylinositol-3-Kinase pathway. By treating HUVEC with the specific tyrosine kinase inhibitor STI571 and over-expressing a dominant negative c-ABL mutant, we show that the VEGF-A-activated c-ABL reduces the amplitude of Mitogen-Activated Protein Kinases (ERK1/2, JNKs and p38) activation in a dose-dependent manner by a negative feedback mechanism. By analysis of the adaptor proteins NCK1 and GRB2 mutants we further show that the negative loop on p38 is mediated by c-ABL phosphorylation at tyrosine 105 of the adaptor protein NCK1, while the phosphorylation at tyrosine 209 of GRB2 down-modulates ERK1/2 and JNKs signaling. These findings suggest that c-ABL function is to establish a correct and tightly controlled response of endothelial cells to VEGF-A during the angiogenic process.     

Inhibition of mini-TyrRS-induced angiogenesis response in endothelial cells by VE-cadherin-dependent mini-TrpRS

To clarify whether a VE-cadherin-dependent pathway allows mini-TrpRS to inhibit mini-TrpRS-induced new blood vessel formation in endothelial cells (ECs), the inhibitory effects of mutant mini-TrpRS and VE-cadherin on mini-TrpRS-induced angiogenesis were investigated. The effects of mini-TyrRS and mini-TrpRS on EC proliferation were evaluated using an MTT colorimetric assay. Cell migration was assayed using a modified Boyden chamber technique. The angiogenic activity in vitro was evaluated by transwell migration assay and matrigel-induced capillary tube formation. It was found that mini-TrpRS does not inhibit the mini-TyrRS-induced proliferation and migration of EC under the condition of VE-cadherin knockout. While wild-type mini-TrpRS inhibited mini-TyrRS-induced angiogenesis, this activity vanished for the mutant protein. Also, the promotion of angiogenesis by mini-TyrRS and the inhibition of angiogenesis by mini-TrpRS were VEGFR2 dependent but not VEGF dependent. Mini-TyrRS was able to increase the protein expression of VEGFR-2 in the presence of VE-cadherin, while no stimulatory effect of mini-TyrRS was detected when VE-cadherin was not present. Angiogenesis is therefore stimulated by mini-TyrRS and inhibited by mini-TrpRS, raising the possibility that mini-TyrRS and mini-TrpRS stimulate a common downstream signaling event: VE-cadherin. Thus, naturally occurring fragments of the two proteins involved in translation, TyrRS and TrpRS, have opposing activities on angiogenesis. The opposing activities of the two tRNA synthetases suggest tight regulation of the balance between pro- and antiangiogenic stimuli.     

VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses

Optimal angiogenic and lymphangiogenic gene therapy requires knowledge of the best growth factors for each purpose. We studied the therapeutic potential of human vascular endothelial growth factor (VEGF) family members VEGF-A, VEGF-B, VEGF-C, and VEGF-D as well as a VEGFR-3-specific mutant (VEGF-C156S) using adenoviral gene transfer in rabbit hindlimb skeletal muscle. The significance of proteolytic processing of VEGF-D was explored using adenoviruses encoding either full-length or mature (DeltaNDeltaC) VEGF-D. Adenoviruses expressing potent VEGFR-2 ligands, VEGF-A and VEGF-DDeltaNDeltaC, induced the strongest angiogenesis and vascular permeability effects as assessed by capillary vessel and perfusion measurements, modified Miles assay, and MRI. The most significant feature of angiogenesis induced by both VEGF-A and VEGF-DDeltaNDeltaC was a remarkable enlargement of microvessels with efficient recruitment of pericytes suggesting formation of arterioles or venules. VEGF-A also moderately increased capillary density and created glomeruloid bodies, clusters of tortuous vessels, whereas VEGF-DDeltaNDeltaC-induced angiogenesis was more diffuse. Vascular smooth muscle cell proliferation occurred in regions with increased plasma protein extravasation, indicating that arteriogenesis may be promoted by VEGF-A and VEGF-DDeltaNDeltaC. Full-length VEGF-C and VEGF-D induced predominantly and the selective VEGFR-3 ligand VEGF-C156S exclusively lymphangiogenesis. Unlike angiogenesis, lymphangiogenesis was not dependent on nitric oxide. The VEGFR-1 ligand VEGF-B did not promote either angiogenesis or lymphangiogenesis. Finally, we found a positive correlation between capillary size and vascular permeability. This study compares, for the first time, angiogenesis and lymphangiogenesis induced by gene transfer of different human VEGFs, and shows that VEGF-D is the most potent member when delivered via an adenoviral vector into skeletal muscle.     

Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A

During bone formation and fracture healing there is a cross-talk between endothelial cells and osteoblasts. We previously showed that vascular endothelial growth factor A (VEGF-A) might be an important factor in this cross-talk, as osteoblast-like cells produce this angiogenic factor in a differentiation-dependent manner. Moreover, exogenously added VEGF-A enhances osteoblast differentiation. In the present study we investigated, given the coupling between angiogenesis and bone formation, whether bone morphogenetic proteins (BMPs) stimulate osteoblastogenesis and angiogenesis through the production of VEGF-A. For this we used the murine preosteoblast-like cell line KS483, which forms mineralized nodules in vitro, and an angiogenesis assay comprising 17-d-old fetal mouse bone explants that have the ability to form tube-like structures in vitro. Treatment of KS483 cells with BMP-2, -4, and -6 enhanced nodule formation, osteocalcin mRNA expression, and subsequent mineralization after 18 d of culture. This was accompanied by a dose-dependent increase in VEGF-A protein levels throughout the culture period. BMP-induced osteoblast differentiation, however, was independent of VEGF-A, as blocking VEGF-A activity by a VEGF-A antibody or a VEGF receptor 2 tyrosine kinase inhibitor did not affect BMP-induced mineralization. To investigate whether BMPs stimulate angiogenesis through VEGF-A, BMPs were assayed for their angiogenic activity. Treatment of bone explants with BMPs enhanced angiogenesis. This was inhibited by soluble BMP receptor 1A or noggin. In the presence of a VEGF-A antibody, both unstimulated and BMP-stimulated angiogenesis were arrested. Conditioned media of KS483 cells treated with BMPs also induced a strong angiogenic response, which was blocked by antimouse VEGF-A but not by noggin. These effects were specific for BMPs, as TGF beta inhibited osteoblast differentiation and angiogenesis while stimulating VEGF-A production. These findings indicate that BMPs stimulate angiogenesis through the production of VEGF-A by osteoblasts. In conclusion, VEGF-A produced by osteoblasts in response to BMPs is not involved in osteoblast differentiation, but couples angiogenesis to bone formation.     

Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab

Pharmacological inhibition of VEGF-A has proven to be effective in inhibiting angiogenesis and vascular leak associated with cancers and various eye diseases. However, little information is currently available on the binding kinetics and relative biological activity of various VEGF inhibitors. Therefore, we have evaluated the binding kinetics of two anti-VEGF antibodies, ranibizumab and bevacizumab, and VEGF Trap (also known as aflibercept), a novel type of soluble decoy receptor, with substantially higher affinity than conventional soluble VEGF receptors. VEGF Trap bound to all isoforms of human VEGF-A tested with subpicomolar affinity. Ranibizumab and bevacizumab also bound human VEGF-A, but with markedly lower affinity. The association rate for VEGF Trap binding to VEGF-A was orders of magnitude faster than that measured for bevacizumab and ranibizumab. Similarly, in cell-based bioassays, VEGF Trap inhibited the activation of VEGFR1 and VEGFR2, as well as VEGF-A induced calcium mobilization and migration in human endothelial cells more potently than ranibizumab or bevacizumab. Only VEGF Trap bound human PlGF and VEGF-B, and inhibited VEGFR1 activation and HUVEC migration induced by PlGF. These data differentiate VEGF Trap from ranibizumab and bevacizumab in terms of its markedly higher affinity for VEGF-A, as well as its ability to bind VEGF-B and PlGF.     

VEGF-A,HGF and bFGF are involved in IL-17A-mediated migration and capillary-like vessel formation of vascular endothelial cells

Background: Interleukin (IL)-17A possesses biological activities to promote vascular endothelial cell migration and microvessel development. Objective: To clarify which angiogenic factors are involved in IL-17A-modified angiogenesis-related functions of vascular endothelial cell migration and microtube development or not. Methods: The potential contribution of various angiogenic stimulators to in vitro angiogenic activities of IL-17A was assessed with both modified Boyden Chemotaxicell chamber assay and in vitro angiogenesis assay. Results: The addition of a neutralizing antibody (Ab) for hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF)-A to the upper and lower compartments in a modified Boyden Chemotaxicell chamber significantly attenuated human dermal microvascular endothelial cell (HMVEC) migration elicited by IL-17A. Moreover, IL-17A-induced capillary-like microvessel development in human umbilical vein endothelial cell (HUVEC) and human dermal fibroblast (HDF) co-culture system was significantly impaired by a neutralizing Ab against HGF, bFGF, VEGF-A, cysteine-x-cysteine ligand 8 (CXCL8)/IL-8 or cysteine-x-cysteine (CXC) chemokine receptor (CXCR)-2. Conclusion: Our findings demonstrate the involvement of HGF, bFGF, VEGF-A and/or CXCL8/IL-8, to various degrees, in migration and microvessel development of vascular endothelial cells mediated by IL-17A.     

Regulation of multiple angiogenic pathways by Dll4 and Notch in human umbilical vein endothelial cells

The Notch ligand, Dll4, is essential for angiogenesis during embryonic vascular development and is involved in tumour angiogenesis. Several recent publications demonstrated that blockade of Dll4 signalling inhibits tumour growth, suggesting that it may constitute a good candidate for anti-cancer therapy. In order to understand the role of Dll4 at the cellular level, we performed an analysis of Dll4-regulated genes in HUVECs. The genes identified included several angiogenic signalling pathways, such as VEGF, FGF and HGF. In particular we identified downregulation (VEGFR2, placenta growth factor PlGF) of VEGF pathway components resulting in the overall effect of limiting the response of HUVEC to VEGF. However extensive upregulation of VEGFR1 was observed allowing continued response to its ligand PlGF but the soluble form of the VEGFR1, sVEGFR1 was also upregulated. PlGF enhanced tubulogenesis of HUVEC suggesting that downregulation of PlGF and upregulation of VEGFR1 including sVEGFR1 are important mechanisms by which Dll4 attenuates PlGF and VEGF signalling. Dll4-stimulated HUVECs had impaired ERK activation in response to VEGF and HGF indicating that Dll4 signalling negatively regulates these pathways. Dll4 expression reduced vessel sprout length in a 3D tubulogenesis assay confirming that Dll4 signalling inhibits angiogenesis. Altogether, our data suggest that Dll4 expression acts as a switch from the proliferative phase of angiogenesis to the maturation and stabilisation phase by blocking endothelial cell proliferation and allowing induction of a more mature, differentiated phenotype. The regulation of sVEGFR1 provides a novel mechanism for Dll4 signalling to regulate cells at distance, not just in adjacent cells.     

Vascular endothelial growth factor receptor-1 and receptor-2 initiate a phosphatidylinositide 3-kinase-dependent clonogenic response in acute myeloid leukemia cells

OBJECTIVE: Vascular endothelial growth factor (VEGF) interacts with two high-affinity receptor tyrosine kinases (RTK) on vascular endothelium to initiate complementary but disparate biologic responses. We previously reported that acute myeloid leukemia (AML) cells express VEGF and one or both VEGF-A receptors, Flt-1 (VEGFR-1) and KDR (VEGFR-2). To evaluate receptor-selective trophic response to VEGF-A in AML cells, we investigated receptor-specific ligand activation responsible for VEGF-initiated clonogenic response. MATERIALS AND METHODS: Using KG1 (VEGFR-1+/VEGFR-2+) and HL60 (VEGFR-1+) cells with differential VEGF receptor display, we investigated ligand-induced clonogenic response and receptor-initiated signaling after stimulation with VEGF-A, the VEGFR-1 selective ligand placental growth factor (PlGF), or receptor-specific antibody agonists. RESULTS: Recombinant human (rhu)-VEGF increased S-phase fraction and stimulated colony formation in both KG1 and HL60 cells. Ligation of VEGFR-1 or VEGFR-2 with receptor-specific antibody agonists triggered equivalent and concentration-dependent stimulation of colony recovery in KG1 cells, whereas clonogenic response in HL60 cells was restricted to VEGFR-1 activation by antibody or PlGF. In serum-deprived KG1 and HL60 cells, rhu-VEGF stimulated rapid and sustained phosphorylation of Akt/PKB that was inhibited by the phosphatidyl inositol 3-kinase (PI3-K) kinase inhibitor wortmannin. Preincubation with wortmannin inhibited VEGF-induced colony formation in a concentration-dependent fashion. rhu-VEGF-induced clonogenic response and Akt phosphorylation was abolished by the VEGF-RTK inhibitor SU-5416 at concentrations greater than 10 microM, whereas MEK inhibition by PD98059 (1 and 10 microM) was ineffective. In vivo suppression of Akt phosphorylation was confirmed in myeloblast lysates from three patients with advanced myeloid malignancies treated with SU5416. CONCLUSION: These data indicate that VEGF interaction with either VEGFR-1 or VEGFR-2 initiates a clonogenic response in AML cells that is PI3-kinase dependent. RTK inhibitors with broad specificity for angiogenic receptors represent novel therapeutics that merit further clinical investigation in AML.     

Eicosanoid regulation of angiogenesis: role of endothelial arachidonate 12-lipoxygenase

Angiogenesis, the formation of new capillaries from preexisting blood vessels, is a multistep, highly orchestrated process involving vessel sprouting, endothelial cell migration, proliferation, tube differentiation, and survival. Eicosanoids, arachidonic acid (AA)-derived metabolites, have potent biologic activities on vascular endothelial cells. Endothelial cells can synthesize various eicosanoids, including the 12-lipoxygenase (LOX) product 12(S)-hydroxyeicosatetraenoic acid (HETE). Here we demonstrate that endogenous 12-LOX is involved in endothelial cell angiogenic responses. First, the 12-LOX inhibitor, N-benzyl-N-hydroxy-5-phenylpentanamide (BHPP), reduced endothelial cell proliferation stimulated either by basic fibroblast growth factor (bFGF) or by vascular endothelial growth factor (VEGF). Second, 12-LOX inhibitors blocked VEGF-induced endothelial cell migration, and this blockage could be partially reversed by the addition of 12(S)-HETE. Third, pretreatment of an angiogenic endothelial cell line, RV-ECT, with BHPP significantly inhibited the formation of tubelike/cordlike structures within Matrigel. Fourth, overexpression of 12-LOX in the CD4 endothelial cell line significantly stimulated cell migration and tube differentiation. In agreement with the critical role of 12-LOX in endothelial cell angiogenic responses in vitro, the 12-LOX inhibitor BHPP significantly reduced bFGF-induced angiogenesis in vivo using a Matrigel implantation bioassay. These findings demonstrate that AA metabolism in endothelial cells, especially the 12-LOX pathway, plays a critical role in angiogenesis.     

The discovery of the placental growth factor and its role in angiogenesis: a historical review

The placental growth factor (PlGF) is an angiogenic protein belonging to the vascular endothelial growth factor (VEGF) family, which was discovered in 1991 by an Italian scientist, Maria Graziella Persico. Dr Persico cloned and purified PlGF and determined its structure by crystallography resolution. Furthermore, she identified VEGF receptor-1 (VEGFR-1) as the receptor for PlGF, and in collaboration with Dr Peter Carmeliet in Leuven, she generated evidence that loss of PlGF does not affect development, reproduction, or postnatal life. PlGF is expressed primarily in the placenta and is up-regulated in several pathological conditions, although its role is still controversial. Some data in literature reported that PlGF enhances pathological angiogenesis by initiating a cross-talk between VEGFR-1 and VEGFR-2, whereas other studies did not confirm these findings. Regarding the potential therapeutic employment of PlGF, recent evidence has shown that an anti-PlGF antibody may act as a potent antiangiogenic agent, and that it has the advantage of minor toxicity when combined with anti-VEGF strategies.